HydF, an iron-sulfur protein

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Table of contents

Abbreviations and Acronyms
Statement
Chapter I: Introduction
Section A
1.1 Metalloprotein: metal ions in living systems
1.2 Iron-sulfur cluster proteins
1.3 Biosynthesis of Fe–S clusters
1.4 Hydrogenase enzymes
Section B
1.5 The metal center of [FeFe]-hydrogenases
1.5.1 A protein machinery for [FeFe]-hydrogenase maturation
1.5.2 The Hyd proteins
1.5.3 The HydF protein
1.5.3.1 HydF, a scaffold protein
1.5.3.2 HydF, an iron-sulfur protein
1.5.3.3 HydF, a nucleotide-binding protein
1.5.3.4 Structure of the HydF protein
1.5.4 The HydE protein
1.5.4.1 HydE, a radical-SAM enzyme
1.5.4.2 HydE: a second, non-essential, cluster
1.5.4.3 HydE partners
1.5.4.4 Structure of the HydE protein
1.5.5 The HydG protein
1.5.5.1 HydG catalyzes tyrosine conversion to CO and CN
1.5.5.2 HydG: a radical mechanism
1.5.5.3 HydG: an unprecedented [5Fe-4S] cluster
1.5.5.4 Structure of the HydG protein
1.5.5.5 HydG enzyme mechanism
1.5.6 Mechanism of maturation of [FeFe]-hydrogenases
1.5.7 Redox states of the H-cluster of [FeFe]-hydrogenases
1.5.8 Maturase-free Chemical maturation: a unique technological tool
Section C
1.6 Artificial hydrogenases
1.6.1 Artificial hydrogenases based on synthetic diiron complexes
1.6.1.1 Micelles
1.6.1.2 Dendrimers
1.6.1.3 Polymers
1.6.1.4 Oligo-/poly-saccharides
1.6.1.5 Metal-Organic-Frameworks
1.6.2 Ni-based artificial hydrogenases
1.6.3 Artificial hydrogenases based on synthetic cobalt complexes
Section D
Aim of the project
Results and discussion
Chapter II: The [FeFe]-hydrogenase from Megasphaera elsdenii
2.1 Expression and aerobic purification of MeHydA
2.2 [Fe–S] cluster reconstitution of apo-MeHydA
2.3 Static light scattering of apo-MeHydA
2.4 EPR spectroscopic characterization of the iron-sulfur MeHydA
2.5 Chemical maturation
2.5.1 Incorporation of synthetic [Fe2(adt)(CO)4(CN)2]2– complex
2.5.2 Incorporation of synthetic [Fe2(pdt)(CO)4(CN)2]2– complex
2.6 FTIR and EPR spectroscopic characterization of holo-MeHydA
2.7 Conclusion and future perspectives: biotechnological application?
Chapter III: A truncated form of M. elsdenii [FeFe]-hydrogenase
3.1 Strategy 1: Cys to Ser mutations
3.1.1 Expression and purification of CystoSerMeHydA construct
3.2 Strategy 2: MeH-HydA
3.2.1 Cloning experiment
3.2.2 Expression and aerobic purification of apo-MeH-HydA
3.2.3 [4Fe–4S]H cluster reconstitution of MeH-HydA apoprotein
3.2.4 Spectroscopic characterization of FeS-MeH-HydA
3.2.5 Chemical maturation of FeS-MeH-HydA with [Fe2(adt)(CO)4(CN)2]2- synthetic complex
3.2.5.1 MeH-HydA: new maturation conditions led to higher maturation
3.2.6 FTIR and EPR characterization of chemically maturated MeH-HydA
3.2.7 Conclusion and future perspectives
Chapter IV: The [FeFe]-hydrogenase maturase HydF
4.1 HydF protein from Thermotoga maritima
4.1.1 Expression, purification and iron-sulfur reconstitution of HydF from
Thermotoga maritima
4.1.2 Insertion of synthetic complexes 1 and 2 onto TmHydF: hydrogenase activity
4.1.3 X-ray structure of apo-TmHydF, the apoform of HydF from Thermotoga maritima
4.2 Preparation and characterization of iron-sulfur reconstituted HydF from
Thermosipho melanesiensis and Clostridium Thermocellum
4.2.1 X-ray structure of TmeHydF: a [4Fe–4S] with an unexpected and exchangeable ligand
4.2.2 Preparation and characterization of 1- and 2-TmeHydF hybrids
4.2.3 Hydrogenase activity of 2-TmeHydF hybrid protein
4.2.4 Site-directed mutagenesis of TmeHydF: E305C and E305H
4.3 Conclusion and future perspectives
General conclusions
Chapter V: Materials and Methods
5.1 Biologic material
5.1.1 Competent cells
5.1.2 Plasmids
5.1.3 Growth media
5.1.4 Molecular Biology
5.1.4.2 Transformation
5.1.4.3 Plasmid preparation
5.2 Biochemical methods: Protein expression
5.2.1 TmHydF: HydF from Thermotoga maritima
5.2.2 HydF from Thermosipho melanesiensis (TmeHydF) and Clostridium thermocellum (CtHydF)
5.2.2.1 E305C and E305H TmeHydF mutants
5.2.3 MeHydA: the [FeFe]-hydrogenase from Megasphaera elsdenii
5.2.4 MeH-HydA: the [FeFe]-hydrogenase truncated form of Megasphaera elsdenii
5.2.5 CsdA: the Cysteine Desulfurase from E. coli
5.3 Proteins purification
5.3.1 TmHydF: HydF from Thermotoga maritima
5.3.2 HydF from Thermosipho melanesiensis (TmeHydF) and Clostridium thermocellum (CtHydF)
5.3.2.1 E305C and E305H TmeHydF mutants
5.3.3 CsdA from E. coli.
5.3.4 MeHydA: the [FeFe]-hydrogenase from Megasphaera elsdenii
5.3.5 MeH-HydA: the [FeFe]-hydrogenase truncated form of Megasphaera elsdenii
5.4 Biochemical methods: [Fe–S] cluster reconstitution of apo-proteins
5.4.1 Iron-sulfur reconstitution with 57Fe
5.4.2 [Fe–S] protein preparation for EPR and HYSCORE measurements
5.4.2.1 TmeHydF, E305C and E305H mutants
5.4.2.2 MeHydA and MeH-HydA
5.5 Chemical methods: Insertion of [Fe2(adt/pdt)(CO)4(CN)2]2- onto HydF and HydA
5.5.1 Synthesis of (Et4N)2[Fe2(adt/pdt)(CO)4(CN)2] complexes
5.5.2 Insertion of [Fe2(adt/pdt)(CO)4(CN)2]2- complex onto MeHydA
5.5.2.1 Hox state
5.5.2.2 Hox-CO inhibited state
5.5.2.3 Hred state
5.5.3 Insertion of [Fe2(adt)(CO)4(CN)2]2- complex onto MeH-HydA
5.5.3.1 Hox, Hox-CO and Hred states
5.5.4 Insertion of [Fe2(adt/pdt)(CO)4(CN)2]2- complex onto HydF proteins
5.6 Hydrogenase-like activity
5.6.1 Calibration curve for H2 quantitation
5.6.2 H2 detection via Gas Chromatography of MeHydA and MeH-HydA proteins
5.6.3 H2 detection via a miniaturized Clark-type hydrogen sensor
5.6.3.1 Methyl viologen assay for TmHydF, TmeHydF and MeHydA proteins
5.6.3.2 Photocatalytic H2 production assay driven by Ru(bpy)3 2+
5.7 Determination of protein concentration
5.8 Determination of cofactor concentration
5.8.1 Iron quantitation: Fish method
5.8.2 Sulfur quantitation: Beinert
5.9 Spectroscopic characterization
5.9.1 Uv-Visible spectroscopy
5.9.2 EPR spectroscopy
5.9.3 HYSCORE spectroscopy
5.9.4 FTIR spectroscopy
5.9.5 Mössbauer spectroscopy
5.9.6 X-ray Crystallography
5.9.6.1 Refinement statistics
Acknowledgments